Primary structure and characterization of a non hemorrhagic metalloproteinase with fibrinolytic activity, from the snake venom of Protobothrops tokarensis (Tokara-habu).

A low molecular weight metalloproteinase, named PT-H₂ protease, with fibrinolytic activity, was purified from the venom of Protobothrops tokarensis (Tokara-habu) by gel-filtration using Sephadex G-100, and ion-exchange chromatographies using CM-Sepharose Fast Flow and Mono S HR 5/5. By this procedure, about 85 mg of purified protein were obtained from 1.0 g of P. tokarensis venom. The purified protein showed a single protein band with a molecular weight of about 22.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. The pI of purified protein showed a single band of 6.8. This proteinase showed a strong fibrinolytic activity. Further, this proteinase showed fibrinogenase activity and proteolytic activity against synthetic substrates for matrix metalloproteinase, ADAM-17, and TACE (tumor necrosis factor converting enzyme). These proteolytic activities were inhibited by metalloproteinase inhibitors such as EDTA. PT-H₂ protease consisted of 201 amino acid residues and had a calculated molecular weight of 22,994.7 Da. This protein showed conservation of the Zn²⁺-binding HEXXHXXGXXHD sequence. PT-H₂ protease showed high homology from 51.7 to 99.5% with amino acid sequences of other snake venom metalloproteinases.